DEK1 displays a strong subcellular polarity during Physcomitrella patens 3D growth.

Defective Kernel 1 (DEK1) is genetically at the nexus of the 3D morphogenesis of land plants. We aimed to localize DEK1 in the moss Physcomitrella patens to decipher its function during this process. To detect DEK1 in vivo, we inserted the tdTomato fluorophore into PpDEK1 gene locus. Confocal microscopy coupled with the use of time-gating allowed the precise DEK1 subcellular localization during 3D morphogenesis. DEK1 localization displays a strong polarized signal, as it is restricted to the plasma membrane domain between recently divided cells during the early steps of 3D growth development as well as during the subsequent vegetative growth. The signal furthermore displays a clear developmental pattern because it is only detectable in recently divided and elongating cells. Additionally, DEK1 localization appears to be independent of its calpain domain proteolytic activity. The DEK1 polar subcellular distribution in 3D tissue developing cells defines a functional cellular framework to explain its role in this developmental phase. Also, the observation of DEK1 during spermatogenesis suggests another biological function for this protein in plants. Finally the DEK1-tagged strain generated here provides a biological platform upon which further investigations into 3D developmental processes can be performed.

The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data.

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.

Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins.

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gß and Gγ genes, but no canonical Gα Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gß proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGß2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGß2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.

Large-scale proteome analysis of abscisic acid and ABSCISIC ACID INSENSITIVE3-dependent proteins related to desiccation tolerance in Physcomitrella patens.

Desiccation tolerance is an ancestral feature of land plants and is still retained in non-vascular plants such as bryophytes and some vascular plants. However, except for seeds and spores, this trait is absent in vegetative tissues of vascular plants. Although many studies have focused on understanding the molecular basis underlying desiccation tolerance using transcriptome and proteome approaches, the critical molecular differences between desiccation tolerant plants and non-desiccation plants are still not clear. The moss Physcomitrella patens cannot survive rapid desiccation under laboratory conditions, but if cells of the protonemata are treated by the phytohormone abscisic acid (ABA) prior to desiccation, it can survive 24 h exposure to desiccation and regrow after rehydration. The desiccation tolerance induced by ABA (AiDT) is specific to this hormone, but also depends on a plant transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3). Here we report the comparative proteomic analysis of AiDT between wild type and ABI3 deleted mutant (Δabi3) of P. patens using iTRAQ (Isobaric Tags for Relative and Absolute Quantification). From a total of 1980 unique proteins that we identified, only 16 proteins are significantly altered in Δabi3 compared to wild type after desiccation following ABA treatment. Among this group, three of the four proteins that were severely affected in Δabi3 tissue were Arabidopsis orthologous genes, which were expressed in maturing seeds under the regulation of ABI3. These included a Group 1 late embryogenesis abundant (LEA) protein, a short-chain dehydrogenase, and a desiccation-related protein. Our results suggest that at least three of these proteins expressed in desiccation tolerant cells of both Arabidopsis and the moss are very likely to play important roles in acquisition of desiccation tolerance in land plants. Furthermore, our results suggest that the regulatory machinery of ABA- and ABI3-mediated gene expression for desiccation tolerance might have evolved in ancestral land plants before the separation of bryophytes and vascular plants.

The phenotype of the CRINKLY4 deletion mutant of Physcomitrella patens suggests a broad role in developmental regulation in early land plants.

MAIN CONCLUSIONS: Deletion of the ancestral gene of the land plant multigene family of receptor like kinase CR4 in Physcomitrella patens demonstrates involvement in developmental control of gametophytic and sporophytic organs. The CRINKLY4 (CR4) family of receptor kinases in angiosperms consists of three clades, one including CR4, the CR4-related CCR1 and CCR2, a second including CCR3 and CCR4 family members, and a third and more distant clade. In addition to crinkly leaves in maize, which gave rise to the mutant gene name, CR4 is implicated in ovule, embryo, flower and root development in Arabidopsis thaliana. In root tips of the same species the module including a CLAVATA3/ESR-related protein, an Arabidopsis CR4, a CLAVATA1 and a WUSCHEL-related homeobox 5 (CLE40-ACR4-CLV1-WOX5) is implicated in meristem cell regulation. In embryos and shoots, CR4 acts together with A. thaliana MERISTEM LAYER 1 and PROTODERMAL FACTOR 2 to promote A. thaliana epidermis differentiation. Phylogenetic analysis has demonstrated that early land plants, e.g. mosses carry a single ancestral CR4 gene, together with genes encoding the other members of the CLE40-ACR4-CLV1-WOX5 signaling module. Here we show that CR4 serves as a broad regulator of morphogenesis both in gametophyte phyllids, archegonia and in sporophyte epidermis of the moss Physcomitrella patens. The phenotype of the CR4 deletion mutant in moss provides insight into the role of the ancestral CR4 gene as a regulator of development in early land plants.

Prenylation is required for polar cell elongation, cell adhesion, and differentiation in Physcomitrella patens.

Protein prenylation is required for a variety of growth and developmental processes in flowering plants. Here we report the consequences of loss of function of all known prenylation subunits in the moss Physcomitrella patens. As in Arabidopsis, protein farnesyltransferase and protein geranylgeranyltransferase type I are not required for viability. However, protein geranylgeranyltransferase type I activity is required for cell adhesion, polar cell elongation, and cell differentiation. Loss of protein geranylgeranyltransferase activity results in colonies of round, single-celled organisms that resemble unicellular algae. The loss of protein farnesylation is not as severe but also results in polar cell elongation and differentiation defects. The complete loss of Rab geranylgeranyltransferase activity appears to be lethal in P. patens. Labeling with antibodies to cell wall components support the lack of polarity establishment and the undifferentiated state of geranylgeranyltransferase type I mutant plants. Our results show that prenylated proteins play key roles in P. patens development and differentiation processes.

Genetic analysis of DEFECTIVE KERNEL1 loop function in three-dimensional body patterning in Physcomitrella patens.

DEFECTIVE KERNEL1 (DEK1) of higher plants plays an essential role in position-dependent signaling and consists of a large transmembrane domain (MEM) linked to a protease catalytic domain and a regulatory domain. Here, we show that the postulated sensory Loop of the MEM domain plays an important role in the developmental regulation of DEK1 activity in the moss Physcomitrella patens. Compared with P. patens lacking DEK1 (∆dek1), the dek1∆loop mutant correctly positions the division plane in the bud apical cell. In contrast with an early developmental arrest of ∆dek1 buds, dek1∆loop develops aberrant gametophores lacking expanded phyllids resulting from misregulation of mitotic activity. In contrast with the highly conserved sequence of the protease catalytic domain, the Loop is highly variable in land plants. Functionally, the sequence from Marchantia polymorpha fully complements the dek1∆loop phenotype, whereas sequences from maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) give phenotypes with retarded growth and affected phyllid development. Bioinformatic analysis identifies MEM as a member of the Major Facilitator Superfamily, membrane transporters reacting to stimuli from the external environment. Transcriptome analysis comparing wild-type and ∆dek1 tissues identifies an effect on two groups of transcripts connected to dek1 mutant phenotypes: transcripts related to cell wall remodeling and regulation of the AINTEGUMENTA, PLETHORA, and BABY BOOM2 (APB2) and APB3 transcription factors known to regulate bud initiation. Finally, sequence data support the hypothesis that the advanced charophyte algae that evolved into ancestral land plants lost cytosolic calpains, retaining DEK1 as the sole calpain in the evolving land plant lineage.

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²âº-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.

Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in developing seeds of flowering plants. The moss Physcomitrella patens contains the highly conserved DEK1 gene. Using efficient gene targeting, we generated a precise PpDEK1 deletion (∆dek1), which resulted in normal filamentous growth of protonema. Two distinct mutant phenotypes were observed: an excess of buds on the protonema, and abnormal cell divisions in the emerging buds resulting in developmental arrest and the absence of three-dimensional growth. Overexpression of a complete PpDEK1 cDNA, or the calpain domain of PpDEK1 alone, successfully complements both phenotypes. These results in P. patens demonstrate the morphogenetic importance of the DEK1 protein in the control of oriented cell divisions. As it is not for protonema, it will allow dissection of the structure/function relationships of the different domains of DEK1 using gene targeting in null mutant background.

ABSCISIC ACID INSENSITIVE3 regulates abscisic acid-responsive gene expression with the nuclear factor Y complex through the ACTT-core element in Physcomitrella patens.

The phytohormone ABA and the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3)/VIVIPAROUS 1 (VP1) function in protecting embryos during the desiccation stage of seed development. In a similar signaling pathway, vegetative tissue of the moss Physcomitrella patens survives desiccation by activating downstream genes (e.g. LEA1) in response to ABA and ABI3. We show that the PpLEA1 promoter responds to PpABI3 primarily through the ACTT-core element (5'-TCCACTTGTC-3'), while the ACGT-core ABA-responsive element (ABRE) appears to respond to ABA alone. We also found by yeast-two-hybrid screening that PpABI3A interacts with PpNF-YC1, a subunit of CCAAT box binding factor (CBF)/nuclear factor Y (NF-Y). PpNF-YC1 increased the activation of the PpLEA1 promoter when incubated with PpABI3A, as did NF-YB, NF-YC, and ABI3 from Arabidopsis. This new response element (ACTT) is responsible for activating the ABI3-dependent ABA response pathway cooperatively with the nuclear factor Y (NF-Y) complex. These results further define the regulatory interactions at the transcriptional level for the expression of this network of genes required for drought/desiccation tolerance. This gene regulatory set is in large part conserved between vegetative tissue of bryophytes and seeds of angiosperms and will shed light on the evolution of this pathway in the green plant lineage.

Protection of Telomeres 1 is required for telomere integrity in the moss Physcomitrella patens.

In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.

Identification and functional characterization of two Δ12-fatty acid desaturases associated with essential linoleic acid biosynthesis in Physcomitrella patens.

Two Δ(12)-desaturases associated with the primary steps of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis were successfully cloned from Physcomitrella patens and their functions identified. The open reading frames (ORFs) of PpFAD2-1 and PpFAD2-2 consisted of 1,128 bp and code for 375 amino acids. Their deduced polypeptides showed 62-64 % identity to microsomal Δ(12)-desaturases from other higher plants, and each contained the three histidine clusters typical of the catalytic domains of such enzymes. Yeast cells transformed with plasmid constructs containing PpFAD2-1 or PpFAD2-2 produced an appreciable amount of hexadecadienoic (16:2 Δ(9,12)) and linoleic acids (18:2 Δ(9,12)), not normally present in wild-type yeast cells, indicating that the genes encoded functional Δ(12)-desaturase enzymes. In addition, reduction of the growth temperature from 30 to 15 °C resulted in increased accumulation of unsaturated fatty acid products.

An experimental method to facilitate the identification of hybrid sporophytes in the moss Physcomitrella patens using fluorescent tagged lines.

• The sequencing of the Physcomitrella patens genome, combined with the high frequency of gene targeting in this species, makes it ideal for reverse genetic studies. For forward genetic studies, experimental crosses and genetic analysis of progeny are essential. • Since P. patens is monoicous, producing both male and female gametes on the same gametophore, and undergoing self-fertilization at a high frequency, the identification of crossed sporophytes is difficult. Usually spores from many sporophytes from a mixed culture must be tested for the production of recombinant progeny. • Here, we describe the use of transgenic lines that express a fluorescent transgene constitutively, to provide a direct visual screen for hybrid sporophytes. • We show that segregations in crosses obtained with this technique are as expected, and demonstrate its utility for the study of the rate of outcrossing between three isolates of P. patens.

Mosses as model systems for the study of metabolism and development.

The haploid gametophyte stage of the moss life cycle is amenable to genetic and biochemical studies. Many species can be cultured on simple defined media, where growth is rapid, making them ideal material for metabolic studies. Developmental responses to hormones and to environmental inputs can be studied both at the level of individual cells and in multicellular tissues. The protonemal stage of gametophyte development comprises cell filaments that extend by the serial division of their apical cells, allowing the investigation of the generation and modification of cell polarity and the role of the cytoskeleton in these processes. Molecular techniques including gene inactivation by targeted gene replacement or by RNA interference, together with the nearly completed sequencing of the Physcomitrella patens genome, open the way for detailed study of the functions of genes involved in both development and metabolism.

Physcomitrella patens: mosses enter the genomic age.

The sequenced genome of the moss Physcomitrella patens provides a powerful tool for comparative analyses of land plant genomes. In parallel, several tools for studying gene function have been developed in P. patens, including RNA interference, inducible promoters and gene targeting, a unique attribute of this plant system. The results of these initiatives are now being realized. For example, transcriptomic analyses illustrate commonalities among plant lineages in gene content, structure, and regulation. Transgenic studies show that the regulatory factors ABSCISIC ACID INSENSITIVE3 (ABI3) and LEAFY (LFY) have molecular functions that are conserved between moss and angiosperms, in spite of the fact that they function in non-homologous tissues. Future work in P. patens will contribute to our understanding of the molecular basis of plant development and evolution.

Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss Physcomitrella patens.

We employed a comparative genomic approach to understand protein phosphatase 2C (PP2C)-mediated abscisic acid (ABA) signaling in the moss Physcomitrella patens. Ectopic expression of Arabidopsis (Arabidopsis thaliana) abi1-1, a dominant mutant allele of ABI1 encoding a PP2C involved in the negative regulation of ABA signaling, caused ABA insensitivity of P. patens both in gene expression of late embryogenesis abundant (LEA) genes and in ABA-induced protonemal growth inhibition. The transgenic abi1-1 plants showed decreased ABA-induced freezing tolerance, and decreased tolerance to osmotic stress. Analyses of the P. patens genome revealed that only two (PpABI1A and PpABI1B) PP2C genes were related to ABI1. In the ppabi1a null mutants, ABA-induced expression of LEA genes was elevated, and protonemal growth was inhibited with lower ABA concentration compared to the wild type. Moreover, ABA-induced freezing tolerance of the ppabi1a mutants was markedly enhanced. We provide the genetic evidence that PP2C-mediated ABA signaling is evolutionarily conserved between Arabidopsis and P. patens.

An actinoporin plays a key role in water stress in the moss Physcomitrella patens.

* Modern land plants arose from a green algae-like ancestor c. 480 million years ago. While several novel morphological features were critical for survival in the aerial environment, physiological innovation undoubtedly played a key role in the colonization of terrestrial habitats. Recently, actinoporin genes, a small group of pore-forming toxins from sea anemones, have been found in the bryophyte and lycophyte lineages of land plants where they are upregulated in water-stressed tissues. * The bryoporin gene in the moss Physcomitrella patens (PpBP) was functionally characterized by RNA blot analyses and overexpression in P. patens. In order to examine functional homology between PpBP and sea anemone actinoporins, the recombinant PpBP was subjected to hemolytic analysis of pig blood cells, which is one of the specific activities of actinoporins. * PpBP was upregulated by various abiotic stresses, in particular most strongly by dehydration stress. Overexpression of the bryoporin gene heightens drought tolerance in P. patens significantly. In addition, PpBP shared the highest structural homology with actinoporins in a three-dimensional structural database and showed hemolytic activity. * These results suggest that this phylogenetic distribution may have resulted from an ancient horizontal gene transfer and actinoporins may have played an important role in early land plants.

Production of taxa-4(5),11(12)-diene by transgenic Physcomitrella patens.

Taxadiene synthase gene from Taxus brevifolia was constitutively expressed in the moss Physcomitrella patens using a ubiquitin promoter to produce taxa-4(5),11(12)-diene, the precursor of the anticancer drug paclitaxel. In stable moss transformants, taxa-4(5),11(12)-diene was produced up to 0.05% fresh weight of tissue, without significantly affecting the amounts of the endogenous diterpenoids (ent-kaurene and 16-hydroxykaurane). Unlike higher plants that had been genetically modified to produce taxa-4(5),11(12)-diene, transgenic P. patens did not exhibit growth inhibition due to alteration of diterpenoid metabolic pools. Thus we propose that P. patens is a promising alternative host for the biotechnological production of paclitaxel and its precursors.